Abstract

Resistance to current therapies still impacts a significant number of melanoma patients and can be regulated by epigenetic alterations. Analysis of global cytosine methylation in a cohort of primary melanomas revealed a pattern of early demethylation associated with overexpression of oncogenic transcripts. Loss of methylation and associated overexpression of the CSF 1 receptor (CSF1R) was seen in a majority of tumors and was driven by an alternative, endogenous viral promoter in a subset of samples. CSF1R was particularly elevated in melanomas with BRAF and other MAPK activating mutations. Furthermore, rebound ERK activation after BRAF inhibition was associated with RUNX1-mediated further upregulation of CSF-1R and its ligand IL-34. Importantly, increased CSF-1R and IL-34 overexpression were detected in an independent cohort of resistant melanomas. Inhibition of CSF-1R kinase or decreased CSF-1R expression by RNAi reduced 3-D growth and invasiveness of melanoma cells. Coinhibition of CSF-1R and BRAF resulted in synergistic efficacy in vivo. To our knowledge, our data unveil a previously unknown role for the autocrine-regulated CSF-1R in BRAF V600E resistance and provide a preclinical rationale for targeting this pathway in melanoma.

Figure 7

(A) Using a constant ratio, the V600E inhibitor was combined with either the CSF-1R inhibitor or a MEK inhibitor based on the IC50 value of each drug and used to treat melanoma cells in quadruplicate for 72 hours. The alamarBlue assay was then performed to estimate cell growth inhibition, and curves (mean ± SEM) are shown with the combination index (CI) indicated in the table below for each condition where values <1 demonstrate synergy. (B) Micrographs taken on the fifth day of A2058 3-D cell culture grown with serial dilutions of PLX3397 or PLX4720, as well as of the combination of these 2 inhibitors. Growth inhibition effect of these conditions depicted as median colony size shown. Fifty colonies were measured for each condition. For statistical analysis, see Supplemental Figure 6, D, E, and F. (C) Kaplan-Meier survival curve of A2058 xenografted mice (10 in each group) treated with PLX3397 or PLX4720 or with PLX3397 plus PLX4720 demonstrating significant improvement with combination (log-rank [Mantel-Cox] test, P values indicated in Supplemental Figure 6G). (D) The effect of combined PLX3397/PLX4720 treatment on signaling in the WM-266-4 cell line as depicted by immunoblotting against phospho-ERK, total ERK, phospho-AKT, total AKT, and actin. Cells were lysed after a 96-hour, 3 μM PLX4720 time-course treatment with or without 30 μM PLX3397 added in the last hour. (E) The effect of IL-34 and CSF-1 on the rebound of ERK phosphorylation in A2058 cells as demonstrated by immunoblotting detecting phospho-ERK1/2, total ERK1/2, and actin. Total melanoma cell extract were probed after a 2-hour treatment of 3 μM PLX4720 with or without 30 μM PLX3397, with 100 ng/ml rCSF-1 or rIL-34 added in the last 15 minutes.